Isolation of full-length complementary deoxyribonucleic acid (cDNA) is a key step in the investigation of gene expression, gene function, protein structure, and protein-protein interactions. The polynucleotide amplification process is one of the most important steps of the isolation process. The most common technique for amplifying polynucleotides is the PCR (polymerase chain reaction) method. DNA syntheses by the PCR method involve repetitive complementary chain syntheses in the direction from a primer to the 3′ end. In the PCR method, newly synthesized DNA is utilized as a template after denaturation, and thus the reaction products exponentially increase. Primers used in the PCR reaction comprise nucleotide sequences complementary to the 3′-side region of a desired nucleotide sequence. Accordingly, the nucleotide sequence of at least a region to be annealed by a primer should be known in advance. In other words, DNA of unknown nucleotide sequence cannot be selectively amplified by the PCR method.
However, circumstances exist wherein DNAs of unknown nucleotide sequence need to be selectively amplified. For example, it is often necessary to elicit a full-length gene based only on its partial nucleotide sequence. However, it is particularly difficult to synthesize a cDNA from the 5′-side region of an mRNA. Accordingly, cDNAs having incomplete 5′-side nucleotide sequences are often cloned. To reveal the full-length nucleotide sequence of an incomplete cDNA lacking a 5′-side nucleotide sequence requires the selective amplification of DNAs comprising such unknown 5′-end nucleotide sequences.
In addition, differential display methods, whose goal is to acquire genes having specific functions, often results in the acquisition of nucleotide fragments and partial gene sequences. When the resulting nucleotide sequences do not match with known gene sequences, identification of the entire nucleotide sequence of these genes is required. In such cases, acquisition of full-length genes based on fragmentary nucleotide sequences is attempted.
Methods for acquiring the full-length gene based on a partial gene sequence are known. A typical method is the RACE (rapid amplification of cDNA ends) method. With this method, the region between a known site and each end of an unknown mRNA sequence is amplified (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA 1988 December, 85: 8998-9002). Specifically, the RACE method employs a gene having unknown terminal nucleotide sequence as a template, and amplifies a region containing that unknown nucleotide sequence by PCR using primers directed to a region of known nucleotide sequence. By determining the nucleotide sequence of the PCR products, the unknown nucleotide sequence can be revealed. There are some known variations of the RACE method, including, for example, the SMART (switching mechanism at 5′ end of RNA transcript) method (Nucleic Acids Res. 27/6, 1558-1560, 1999) and the Marathon-ready cDNA method, both supplied by Clontech. Other variations of the RACE method are also known. For example, by ligating both ends to cyclize the DNA, and by using a region of known nucleotide sequence, a PCR primer can be designed even for a DNA having unknown nucleotide sequences at its ends. However, this method lacks specificity for the 5′-end of mRNA. Accordingly, it is particularly unsuitable for acquisition of a full-length gene having unknown 5′-side. Another method involves the linking of an oligonucleotide of known sequence to the 3′-end of a cDNA first strand. However, this method requires a step of purifying the first strand after synthesis.
While the currently known methods have their advantages and have proved to be quite useful, problems still arise. For example, because nonspecific products are often amplified, the specific target cDNA makes up only a small fraction of the total yield. Thus, acquiring the full-length sequence of a gene having low transcription level remains difficult, particularly in circumstances where the mRNA is longer. Accordingly, a new RACE method capable of acquiring full-length sequences based on a very small amount of mRNA would be of particular benefit.